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WT1-origin HLA-DR-binding antigen peptide

一种癌抗原、组合物的技术，应用在HLA-DRB1＊0405-结合抗原肽领域

Active Publication Date: 2009-11-18
INT INST OF CANCER IMMUNOLOGY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports of WT1-derived peptides capable of binding different isoforms

Method used

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  • WT1-origin HLA-DR-binding antigen peptide
  • WT1-origin HLA-DR-binding antigen peptide
  • WT1-origin HLA-DR-binding antigen peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0191] 1. Preparation of Dendritic Cells

[0192] Peripheral blood mononuclear cells (PBMCs) were isolated from blood of HLA-DRB1*0405-positive healthy volunteers by density gradient centrifugation using Ficoll-Paque. Convert the resulting 8x10 6 PBMCs were suspended in 2mlX-VIVO 15 containing 1% AB serum TM culture medium (Camblex), inoculated in a 6-well culture plate, and cultured for 2 hours. After culturing, non-adherent cells were removed and adherent cells were washed with Hanks solution. In X-VIVO15 containing 1% AB serum, 1000U / ml IL-4 and 1000U / ml GM-CSF TM Culture adherent cells in culture medium. On days 2 and 4 of culture, half of the medium was replaced with fresh medium. On day 6, TNF-α was added to a final concentration of 100 U / ml. Cells that survived on day 7 were used as experimental dendritic cells.

[0193] 2. Preparation of CD4 positive T cells (helper T cells)

[0194] Blood obtained from the same healthy volunteer was used as described in (1) ab...

Embodiment 2

[0203] Establishment of CD4-positive T cell lines specific for WT1 peptide

[0204] Dendritic cells prepared by a method similar to Example 1 were prepared in 10 4 Cells / well were seeded in 96-well plates, and then 10 3 Cells / well seeded as WT1 332-347 CD4 positive T cells induced by peptide (SEQ ID NO: 24). As for the medium, X-VIVO 15 containing 1% AB serum, 20 U / ml IL-2 and 5 μg / ml PHA was used TM Medium. The CD-4 positive T cell line was established by continuous culture and named "G2 cell line". The response of the G2 cell line to dendritic cells stimulated with the peptide was determined by a method similar to that of claim 1 . The results are shown in figure 2 middle. When used with WT1 332-347 The G2 cell line showed a proliferative response when co-cultured with peptide-stimulated dendritic cells but not when co-cultured with non-peptide-stimulated dendritic cells.

[0205] These results suggest that the G2 cell line is a WT1-specific 332-347 Peptide CD4 ...

Embodiment 3

[0207] WT1 peptide antigen presentation to HLA-DR molecules

[0208] In a similar manner to Example 1, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of HLA-DRB1*0405-positive healthy volunteers by density gradient centrifugation using Ficoll-Paque. Then, with 10 7 Cells / well PBMCs were seeded in 24-well plates. As the medium, RPMI1640 medium containing 10% FCS and 55 µM 2ME was used. After addition of a medium containing Epstein-Barr virus (EBV), culture was continued for another 4 weeks to establish a B-cell line transformed with EBV, which was named "B-LCL(-) cells". EBV was prepared from the culture supernatant of B95-8 (JCRB Cell Bank No. 9123), which is an EBV-producing cell line. Adjust B-LCL(-) cells to 3x10 7 cells / mL, and a medium containing a virus expressing the WT1 gene was added thereto, followed by addition of polypropylene (final concentration, 8 μg / mL), and the mixture was added to a 24-well plate at 1 ml / well. After culturing f...

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Abstract

The present invention provides a WT1-derived HLA-DRB1*0405-binding antigen peptide, a polynucleotide encoding said peptide, a helper T cell inducer comprising said peptide or polynucleotide, and the like. It is related to a partial peptide consisting of 10 - 25 contiguous amino acids in the amino acid sequence of human WT1 shown in SEQ ID NO: 1, which binds to HLA-DRB1*0405 and induces helper T cells, a polynucleotide encoding said peptide, or a helper T cell inducer comprising said peptide or polynucleotide.

Description

[0001] This application is a divisional application of the following application: filing date November 4, 2004, application number 200480039886.5 (PCT / JP2004 / 016336), title "WT1-derived HLA-DR-binding antigen peptide". technical field [0002] The present invention relates to WT1-derived HLA-DRB1*0405-binding antigen peptides. Background technique [0003] The WT1 gene (Wilms' tumor gene 1) has been identified as one of the causative genes of Wilms' tumor, ie, pediatric renal tumor (Cell 60:509, 1990, Nature 343:774, 1990). The WT1 gene encodes the transcription factor WT1, which plays an important role in many processes such as cell proliferation, differentiation, apoptosis and tissue development (Int. Rev. Cytol. 181: 151, 1998). Originally the WT1 gene was described as a tumor suppressor gene. However, follow-up studies revealed that the WT1 gene is highly expressed in leukemia and a variety of solid tumors including lung and breast cancers, indicating that the WT1 gene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12C12N15/63C12N5/10C12P21/02C07K16/32A61K39/00A61P35/00A61K38/00A61P37/04C07K14/47C07K14/82C07K19/00C12N1/15C12N1/19C12N1/21C12N5/12C12N15/09
CPCC07K14/4748A61K39/0011A61K38/00A61P35/00A61P37/04A61K39/001153C07K14/47C12N15/11
Inventor 杉山治夫
Owner INT INST OF CANCER IMMUNOLOGY INC
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